This invention is directed to a method of inhibiting Phospholipase D. It is further directed to the use of Phospholipase D inhibitors to inhibit Phospholipase D-mediated degradation of membrane phospholipids in plants and produce.
The maturation and ripening of several perishable plant produce is associated with the catabolic breakdown of cellular structures such as membrane and cell wall which is a normal process in the development of ideal organoleptic quality. Untimely destruction of the cellular integrity of produce as occurs during processing (cutting, wounding, blending, maceration etc.) or storage (injury due to chilling) can lead to accelerated destruction of cellular structures sometimes resulting in the loss of quality of the product.
Phospholipase D (PLD) is a ubiquitous, key enzyme that catalyzes the hydrolysis of membrane phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, etc . . . to yield phosphatidic acid and the respective headgroup (Kates, 1955; Galliard, 1980; Exton, 1997). In vitro, PLD also catalyzes the exchange of phospholipid headgroups with primary alcohols such as methanol and ethanol, leading to the formation of phosphatidylalcohols. This reaction is termed transphosphatidylation (Galliard, 1980; Cockcroft, 1997). PLD is wide-spread in a variety of plant species (Quarls and Dawson, 1969; Galliard, 1980). Soluble and membrane-associated PLD have been reported in various plant species (Yoshida, 1979; Galliard, 1980; Xu et al., 1996). Various physiological processes such as germination (Wang et al., 1993), growth of seedlings (Herman and Chrispeels, 1980), stress-induced changes and senescence (Yoshida, 1979; Paliyath and Droillard, 1992) have been suggested to be regulated by PLD. A high activity of PLD has been found in storage tissues, especially in seeds (Heller et al., 1974). Activation of PLD is elicited by a variety of agonists in different cell types leading to the tandem generation of messengers, namely phosphatidic acid and diacylglycerol, that affect many significant. cellular processes (Paliyath and Droillard, 1992; Exton, 1997).
Previous studies have shown that exposure of black locust bark tissues to frost as well as wounding (Yoshida, 1979) and chilling of maize seedlings (Pinhero et al., 1998) resulted in an increase in PLD activity causing massive lipid degradation and membrane deterioration. It has also been reported that PLD is responsible for the hydrolysis of spherosome membrane phospholipids which causes triacylglycerol leakage from spherosomes with subsequent degradation (Takano et al., 1989). Increased association of PLD with microsomal membranes has been proposed to promote PLD-mediated degradation of membrane lipids during xcex3-irradiation and senescence (Voisine et al., 1993; Ryu and Wang, 1995). During fruit ripening, decreased fluidity of microsomal membrane has been reported to activate PLD and increase membrane catabolism (McCormac et al., 1993). Under such conditions PLD activity proceeds in an autocatalytic fashion leading to the total destruction of structural and functional organization of the membrane and abolition of membrane compartmentation (Paliyath and Droillard, 1992).
Preservation of membrane compartmentation is a must for maintaining th e quality of unprocessed perishable plant produce such as corn kernels, leafy vegetables, flowers (cauliflower, broccoli) and fruit. Many of these commodities are used for fresh consumption as well as processing, such as canning, making of soups, jams, sauces, and blending for juice-making. During processing, various produce are subjected to chilling, freezing, heating, mixing with salts, preservatives, solvents etc. which destroy the cellular compartmentation. Excessive catabolic break down can lead to loss of processed food quality such as that observed in the mushiness and off flavour of some processed products. PLD activity appears to be regulated by a number of factors including temperature. To inhibit PLD activity processing corn is harvested in bulk and stored at low temperature. Chilling of corn should be rapid to quickly bypass the temperature regime between 15xc2x0 C. and 5xc2x0 C. where the activity of PLD is much higher. However, at lower temperatures, there is also differential stimulation of other enzymes involved in membrane lipid degradation. For instance, phosphatidate phosphatase and lipolytic acyl hydrolase activities are considerably lower at 4xc2x0 C. than PLD activity in tomato microsomal membranes (Todd et al. 1992 ). If this were to occur in vivo, there would be abundant accumulation of phosphatidic acid in the membrane at low chilling temperatures that would lead to break down in cellular compartmentation. This would decrease the quality of the produce. Consequently, a product and method for preventing membrane breakdown is required.
The present invention provides a method of inhibiting phospholipase D mediated degradation of membrane phospholipids by administering to the membrane an effective amount of a phospholipase D inhibitor to inhibit degradation. In one embodiment the phospholipase D inhibitor is administered to tissue comprising the membrane. In a preferred embodiment of the invention, the phospholipase D inhibitor is selected from the group consisting of C6 aldehydes, C6 alcohols, and monoterpene aldehydes and alcohols. Examples of suitable monoterpene alcohols and aldehydes include: geraniol, citronellol, nerol, and their corresponding aldehydes. More preferably, the phospholipase D inhibitor is hexanal, hexanol, hexenal or hexenol. Most: preferably the phospholipase D inhibitor is hexanal or hexanol.
Preferably, the membrane is a membrane of a plant, fruit or vegetable and the phospholipase D inhibitor is administered to the plant, fruit or vegetable. In a preferred embodiment the membrane is that of sweet corn (Zea mays). In another embodiment the membrane is that of tomato fruit.
In another aspect of the invention, there is provided a method of inhibiting maturation or ripening of a plant, fruit or vegetable, comprising administering to the plant, fruit or vegetable an effective amount of a phospholipase D inhibitor. Preferably, the inhibitor is selected from the group consisting of C6 aldehydes, C6 alcohols, and monoterpene aldehydes and alcohols. Examples of suitable monoterpene alcohols and aldehydes include geraniol, citronellol, nerol, and their corresponding aldehydes. More preferably the phospholipase D inhibitor is hexanal, hexanol, hexenal or hexenol. Most preferably the phospholipase D inhibitor is hexanal or hexanol.
In another embodiment of the invention, there is provided a use of an effective amount of a phospholipase D inhibitor for inhibiting phospholipase D-mediated degradation of membrane phospholipids. Preferably, the inhibitor is selected from the group consisting of C6 aldehydes and C6 alcohols and monoterpene aldehydes and alcohols. Examples of suitable monoterpene alcohols and aldehydes include geraniol, citronellol, nerol, and their corresponding aldehydes. More preferably the phospholipase D inhibitor is hexanal, hexanol, hexenal or hexenol. Most preferably the phospholipase D inhibitor is hexanal or hexanol.
In one embodiment, the invention is directed to a plant, fruit, or vegetable that has been treated with a phospholipase D inhibitor, preferably, in an amount effective to inhibit maturation or ripening of the plant, fruit or vegetable. The phospholipase D inhibitor is preferably selected from the group consisting of C6 aldehydes and C6 alcohols and monoterpene aldehydes and alcohols. Examples of suitable monoterpene alcohols and aldehydes include geraniol, citronellol, nerol, and their corresponding aldehydes. More preferably the phospholipase D inhibitor is hexanal, hexanol, hexenal or hexenol. Most preferably the phospholipase D inhibitor is hexanal or hexanol.
The term xe2x80x9ceffective amountxe2x80x9d as used herein would be understood by a person skilled in the art when reading the present description to mean an amount which is sufficient to produce the specified desired result, whether it be inhibition of Phospholipase D activity, inhibition of membrane phospholipid degradation, or the inhibition of maturation or ripening of a plant, fruit or vegetable. Preferably the phospholipase inhibitor is applied at a concentration of 0.01-01 xcexcl/ml if it is to be left on the membrane. In another preferred embodiment the inhibitor is applied at a concentration of 1-10 xcexcl/ml, potentially as a sprayed 1xc3x97 pulse, and subsequently washed off after treatment.
The term xe2x80x9ctissuexe2x80x9d as used herein means a mass of similar cells and their intercellular substance, working together to perform a particular function.
In an embodiment the inhibitor is administered to the membrane or tissue comprising the membrane in a non-toxic amount. That is an amount which in the end product is not-toxic or not harmful to the end user of the product.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.